A vaccine comprising a protein or its partial peptide derived from a microorganism (a “subunit vaccine”) is advantageous since it can be conveniently prepared through chemical synthesis or recombinant techniques and may have superior safety to live vaccines or inactivated vaccines. However, such subunit vaccines tend to exhibit a lower immunostimulating activity than live vaccine or inactivated vaccine alternatives. In order to improve the immunostimulating activity of subunit vaccines, they may be administered with a vaccine adjuvant in combination with an antigen.
A vaccine adjuvant is an additive enhancing the mammalian immune response and/or cellular immunity response to an antigen. Alum, saponin and the like are used as vaccine adjuvants.
Recently, it has been found that Toll-like Receptors (“TLRs”) play an important role in activating innate immunity, which is one of the common host defense mechanisms against microorganisms. Immune modifying agents such as monophosphoryl lipid A (MPL), CPG ODN and the like can exhibit immunostimulating activity via TLRs.
Amongst the 13 known TLRs identified in human beings several are associated with the recognition of bacterial components (TLRs 1, 2, 4, 5, and 6), viral RNA (TLRs 3, 7, and 8) or unmethylated DNA (TLR 9) (see, Non-patent Reference 1).
It is known that TLR 7 and TLR 8 activators include low molecular weight mimics of the viral single-stranded RNA which is a natural ligand for the receptors in question. For example, synthetic compounds such as 8-oxoadenine compounds (see, Patent References 1, 2, and 3) and imidazoquinoline compounds (see, Patent Reference 4) which mimic viral RNA have been already reported to activate TLR 7 and/or TLR 8.
When TLR 7 and/or TLR 8 are activated, Th1 cells are induced via a TLR/MyD88-dependent signal transduction pathway to activate dendritic cells (DCs). Consequently, the expression of T cell co-stimulatory molecules (CD80, CD86, and CD40) is enhanced and inflammatory cytokines including interferon type I (particularly IFNα), IFNγ, TNFα, IL-6 or IL-12 are produced.
It is also known that TLR 7 and/or TLR 8 activators trigger B cells and further stimulate NK cells to promote IFNγ production as well as DC activation. These pathways are expected to contribute to vaccine adjuvant activity. Indeed, the adjuvant activity of TLR 7/TLR 8 activators such as Resiquimod and Imiquimod has already been reported (see, Non-patent References 2 and 3).
Nevertheless, it is desirable to develop a novel vaccine adjuvant to activate TLR 7 and/or TLR 8.
Squalene is an oily substance used as an ingredient in oil-in-water or water-in-oil emulsion formulations. It is known to enhance the immunostimulating activity of an antigen when used as a surfactant in water-in-oil or oil-in-water emulsions associated with an antigen. Indeed, squalene is used as a base substance of the known vaccine adjuvant MF59, which is useful as an influenza vaccine (see, Non-patent References 4, 5, and 6).
Complexes of TLR 7 and/or TLR8 activator and another substance are known. For example, a vaccine adjuvant prepared by covalently-binding a fatty acid and an imidazoquinoline compound has been reported to allow a TLR 7 activator to be localized in the target tissue, reducing the metabolism and toxicity of the TLR 7 activator (see, Patent References 5, 6, and 7, and Non-patent Reference 7) Furthermore, a complex of a fatty acid glyceride and an imidazoquinoline compound (see, Patent Reference 8), a complex of a fatty acid glyceride and an adenine compound (see, Patent Reference 9), and a complex of phospholipid and an adenine compound (see, Patent Reference 10) are known. A complex of a fatty acid glyceride and an adenine compound via polyethylene glycol has also been reported (see, Patent Reference 11).
However, no complexes of a TLR 7/8 activator and squalene have yet been reported.